This project is a study of the mechanism of ATP-dependent surface contour smoothing in the echinocyte-discocyte transformation of the human erythrocyte membrane. Aspects of this problem will be studied at the level of the intact cell, isolated membrane, inside-out vesicle, isolated membrane skeleton, and detergent-solubilized membrane proteins. Vanadate, thiophosphates and other agents previously show to inhibit ATP-dependent shape changes of members will be applied to intact cells. ATP-dependent transbilayer movements of flourescent aminophospholipids will be studied in inside-out vesicles from cells labeled at the external surgace with flourescent phospholipip analogs. Novel methods based on affinity isolation after chemical modification and fluorescence resonance energy transfer will be developed to analyze the properties of the aminophospholipid translocating system in siolated inside-out vesicles. These methods will be used in studying the effect of membrane skeleton components on the vesicle bilayer and the effect of vesicles bound to intact membrane skeletons. The vanadate-sensitive MgATPase of the membrane will be partially purified after detergent solubilization. Its role in aminophospholipid movements will be evaluated by characterizing its effect on bilayer asymmetry when reconstituted into liposomes containing fluorescent phospholipid analogs. The overall objective of the research is to elucidate the mechanism(s) by which erythrocytes actively maintain the smooth biconcave shape characteristic of their normal viable state.